Vitamin K1) in Olive Oils

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Analytical Determination of Phylloquinone (Vitamin K1) in Olive Oils. Comparison with Other Vegetable Oils Catherine Rebufa, Jacques Artaud

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Catherine Rebufa, Jacques Artaud. Analytical Determination of Phylloquinone (Vitamin K1) in Olive Oils. Comparison with Other Vegetable Oils. European Journal of Lipid Science and Technology, Wiley-VCH Verlag, 2018, 120 (6), 10.1002/ejlt.201700527. hal-01928770

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Phylloquinone www.ejlst.com Analytical Determination of Phylloquinone (Vitamin K1) in Olive Oils. Comparison with Other Vegetable Oils

Catherine Rebufa* and Jacques Artaud

have different cofactors (as the conversion of Olive oil is mainly consumed in the Mediterranean basin and is an important specific peptide-bound glutamate (Glu) source of lipids, antioxidants, and vitamins. Vitamin E (tocopherols) and residues to γ-carboxyglutamate (Gla)) or activities and multiple functions according phylloquinone (vitamin K1), are present in oils. If vitamin E is the subject of to processes (absorption, transport, cellular numerous studies, it is not the case for phylloquinone. The aim of this work uptake, tissue distribution, and turnover), – is to uncover the latest advancements on phylloquinone contents in olive and developed in detail in four reviews.[1 4] vegetable oils. A bibliometric study, from Google Scholar and Web of Science Adequate intake of vitamin K is recom- databases, on the determination of phylloquinone content in vegetable oils mended for all ages and gender people made it possible to count a large number of scientific papers related to food (infants, children, pregnant, and breastfeed- ing women and men); it ranges from 55 matrices but few articles on olive and vegetable oils. The analysis of relevant to 90 μgday 1 for adult women and – works allows the comparison of the phylloquinone content of olive oils to the 65–120 μgday 1 for adult men.[4 6] No other vegetable oils. The different steps of oil sample preparation before their tolerable upper limit has been defined. analysis are reviewed. A compilation of analytical conditions and methods is Nevertheless, the consumption of various realized and it is be found that liquid chromatography with post reduction dietary supplements or food rich on PH column and fluorescence detection is the technique most appropriate. On must be reduced when people take anticoagulant medication in order to avoid any the basis of their phylloquinone content, two oil groups are highlighted; olive adverse outcomes.[6] The major dietary oil belongs to the oil groups (canola, soybean, pumkin, avocado, and source of vitamin K is phylloquinone within 1 cottonseed) having higher values (60–348 μg 100 g ) of phylloquinone. the chloroplasts of green plants.[7] Also, Pratical Application: Phylloquinone (or vitamin K1) content in vegetable oils different phylloquinone databases from and particularly in olive oils is little documented. Phylloquinone exists under E food matrix (in USA, UK, Netherlands, [8–12] and Z forms in oils. The recommended daily intake (for women and men) varies and Japan) have been published. These works showed that the PH intakes came μ 1 between 55 and 120 g day for patients without anticoagulant medication. largely from leafy vegetables (as Broccoli, The knowledge of the two isomers content in vegetable oils is important in cabbage,Perilla,spinach...)whereits content nutrition and heath fields because only the E isomer is bioactive. varied between 113 and 400 μg100g 1.[9,13] Of course, this concentration range depended on cooking process and varietal type of vegetables.[14] Various amounts of PH have been found in algae (green or purple laver, 1. Introduction konbu, hijiki, wakame...)(4–1385 μg100g 1),[12,15] in culinary herbs, dried, or fresh, (Basil, Marjoram, Parsley, Rosemary...) Vitamin K is part of fat-soluble vitamins such as vitamins A, D, and (369–3110 μg100g 1), and spices and seeds (Chilli, Fennel, Green E but it is probably the one that has been the leaststudied. The term pepper, Safron...)(0.125–364 μg100g 1).[16] Variable PH content vitamin K is a generic name, which groups together several has been found in black or green tea leaves compounds having a 2-methyl 1-4-naphthoquinone ring. The (312–1654 μg100g 1) and coffee beans (25 μg 100 g 1)becauseof natural forms of vitamin K (VK) comprise the phylloquinone (PH) the variety,the storage, processing, harvesting and geographic origin (or vitamin K ,VK ) of a vegetable origin and menaquinones MK-n 1 1 but their brews are not an important source of this vitamin (or vitamin K ) of animal origin or bacterial fermentation. Initially 2 (0.03–3.05 μg100g 1) in the same way as certain meats, brewed known for its role in haemostasis, these several molecular forms beverages, soft drink, and alcoholic beverages.[11,12,17] Among the numerous feed habitually eaten bypeople,certainfatsandoils Dr. C. Rebufa, Prof. J. Artaud showed an interested content of PH. PH content of margarines Aix Marseille Univ (defined as product containing not less than 80% fat and derived Univ Avignon from vegetable oils) was depending on the varietal origin of the oils, CNRS, IRD, IMBE margarine processing, and type of margarines (blended, hard, and BEC, F-13013 Marseille, France – μ 1 E-mail: [email protected] soft) (0.4 160 g100g ). But during the hydrogenation process of oil, part of the phylloquinone was transformed into 20, DOI: 10.1002/ejlt.201700527 30 dihydrophylloquinone, compound also analyzed in some

– works.[11,12,18 21] Some mixed dishes contained moderates amounts four isoprenoid units, three of which are reduced. The side chain of phylloquinone that were often attributable to the vegetable oils (also called phytyl chain) with one double bond E (Ia, trans- used in their preparation. Some vegetable oils such as soybean, isomer) or Z (Ib, cis-isomer) is the same phytyl side chain as in cottonseed, rapeseed (canola), and olive were a recognized source of chlorophyll. The coexistence of E and Z isomers was highlighted – PH intakes.[8,11,12,18,19,22 24] Concerning specifically olive oil, two by Cook et al.[20] and Woollard et al.[29] in vegetable oils with Z vitamins were known as minor compounds, the phylloquinone and isomer content relatively high in opposite with those found in vitamin E (general term employed to designate tocopherols and most foods.[5] E isomer is naturally found in all green plants and tocotrienols, including α, β, γ,andδ species). Several works have it is yellow oil soluble in fats (and insoluble in water). It is studied the vitamin E content in olive oil revealing that α-tocopherol commonly designated as phylloquinone or 2-methyl-3-phytyl- δ was the main compound but -tocopherol et tocotrienols have not 1,4-naphthoquinone, or vitamin K1, also K1(20) because of the 20 been detected in olive oil.[25,26] On the other hand, phylloquinone has carbon atoms of its phytyl chain linked on the position 3 on its been the subject of few works because of the small quantities present naphthoquinone ring. Various synonyms are found in different in the olive oil and the difficulties of analyzes of this compound. This articles as phytomenadione (used by The European Pharmaco- review presents the latest advancements on PH content in olive oils. poeia and occasionally found in the pharmaceutical and Thedatacomefromabibliometric study that addresses the pharmacological literature), phytonadione (United States Phar- problems of nomenclature, sampling, stability, analytical methods, macopeia), phytylmenadione, 3-phytylmenadione, 2-methyl-3- and PH content in olive oils and compares them to PH content in (3,7,11,15-tetramethyl-2-hexadecenyl)-1,4-naphthalenedione. vegetable oils. The second natural form groups together the multiprenyl menaquinones with a side chain of 20–60 carbon atoms and many unsaturated side chains. These compounds are soluble in n 2. Historic Background fats, like vitamin K1. The menaquinone with isoprenoids units are commonly called vitamin K n and classified according to the Vitamin K was discovered incidentally, in the course of research 2( ) number of prenyl units (3-methyl-but-2-en-1-yl); number being on the metabolism of cholesterol, undertaken in 1929, by a given as suffix: i.e., menaquinone-n abbreviated MK-n (II).[30] Danish nutritional biochemist, Carl Peter Henrik Dam of the Menaquinones only occur in foods of animal origin or foods Polytechnic Institute of Copenhagen, Denmark. He studied the altered by bacterial fermentation. Generally, microorganisms, role of a low-fat diet on the chicks and noticed that it caused them including bacteria from the human intestine and other animal to bleed. In 1935, he identified the vitamin responsible for species, synthesized menaquinones containing from 4 to 13 coagulation and named it “Koagulation Vitamin.” Also the letter isoprenoid units. Several authors[4,7,31] reported that the origin of K came from the German word “Koagulation.” As early as 1936, the menaquinone MK-4 (III) (also called vitamin K or 2- an oily vitamin K was extracted from alfalfa by H. C. P. Dam, and 2(4) methyl-3-geranyl-geranyl-1,4-naphtoquinone, C H O ) was in 1939, the American biochemist Edward Adelbert Doisy of the 31 40 2 not bacterial but this compound was formed by a re-alkylation St. Louis University, Missouri, synthesized it and named it step from menadione (IV) present in animal feeds or was the vitamin K (phylloquinone). The Doisy group also isolated 1 product of tissue-specific conversion directly from dietary another form of vitamin from putrefied fish meal, another phylloquinone. Among the various synthetic forms of vitamin substance with antihemorrhagic activity which he baptized K, one finds the parent compound, the 2-methyl-1,4-naphtho- vitamin K and now known as menaquinone-6. In 1943, the 2 quinone (IV) (or menadione), also called vitamin K (C H O ), Nobel Prize for Physiology or Medicine was awarded to C. P. H. 3 11 8 2 without side-chain, soluble in water, unlike the previous ones. Dam for his discovery of vitamin K and to E. A. Doisy, for his This vitamin K3, as a provitamin, can be alkylated enzymatically discovery of the chemical nature of vitamin K. A detailed [32] [27] to be converted into vitamin K2(4) in animal tissues and has a historical background was made by Suttie. – biological activity 2 3 times greater than the vitamins K1 and K2. There are another synthetic forms of vitamin K as (i) menadiol 3. Vitamin K Designations (V) (formerly known as vitamin K4 or 2-methylnaphthalene-1,4- diol or reduced menadione or dihydrovitamin K3 or vitamin 0 0 Numerous terms are used to describe the different forms of K3H2,C11H10O2); (ii) the compound 2 ,3 -dihydrovitamin K1 (VI) fi β γ vitamins K in scienti c literature and the most commonly ( , -dihydro vitamin K1, or 2-methyl-3-(3,7,11,15-tetramethyl- encountered are not those derive from IUPAC nomenclature. In hexadecyl)-1,4-naphthalenedione, C31H48O2), resulting of the fact, vitamin K is the generic term of a group of fat-soluble reduction of phylloquinone side chain; (iii) the vitamin K1(25) vitamins of natural or synthetic origins, those the chemical (VII) (or 2-methyl-3-(3,7,11,15,19-pentamethyl-2-eicosenyl)-1,4- structure has a common 2-methyl-1,4-naphthoquinone ring, but naphtalenedione, C36H56O2) produced by the substitution of a differs in the length and degree of saturation of their isoprenoid 25-carbon side chain on C3 carbon of menadione (IV); and (iiii) side chain at the 3-position.[1,28] All the chemical compounds different vitamin K analogs with different length of the alkyl cited below are detailed in Table 1. In nature, this 3-substituent side-chain (VIII).[33] Both synthetic forms are commonly used as depends on the organism by which it is synthesized. Two natural internal standards in vitamin K analysis. The last form that can forms of vitamin K have been isolated, phylloquinone (or be evoked was the one form after a PH derivatization step fi vitamin K1) and menaquinones (or vitamin K2). The rst one is (reduction) to make it detectable: the phyllohydroquinone (IX) R R R E the compound [ -[ , -( )]]-2-methyl-3-(3,7,11,15-tetramethyl- (or hydroquinone or dihydrovitamin K1 or 2-methyl-3- 2-hexadecenyl)-1,4-naphthalenedione (Ia) (IUPAC; CAS num- [(2E,7R,11R)-3,7,11,15-tetramethylhexadec-2-en-1-yl]naphtha- ber: 84-80-0, C31H46O2) which has a lateral chain which contains lene-1,4-diol).

Table 1. Nomenclature and structure of Vitamin K.

Molecular Compounds Synonym names formulae Structural formulae Origin

(Ia) Phylloquinone. vitamin K1, 2-methyl- 3-phytyl-1,4- C31H46O2 Vegetal naphtoquinone. [R-[Ra),Ra)-(E)]]-2-methyl-3- (trans) (3,7,11,15-tetramethyl-2- hexadecenyl)-1, 4-naphthalenedione

(Ib) Phylloquinone, vitamin K1, 2-methyl- 3-phytyl-1,4- C31H46O2 Vegetal naphtoquinone, [R-[R,R-(Z)]]-2-methyl-3- (cis) (3,7,11,15-tetramethyl-2-hexadecenyl)- 1,4-naphthalenedione

a) (II) Vitamins K2(n), menaquinone MK-n Microbial

a) (III) Menaquinone (MK-4), vitamin K2(n), C31H40O2 Microbial 2-methyl-3-geranygeranyl-1,4-naphtoquinone

(IV) Menadione, 2-methyl-1,4-naphtoquinone, C11H8O2 Synthetic

vitamin K3

(V) Menadiol vitamin K4, 2-methylnaphthalene-1,4-diol, C11H10O2 Synthetic

reduced menadione, dihydrovitamin K3. Vitamin K3H2

a) 0 0 β γ (VI) 2 ,3 -dihydrovitamin K1, , -dihydro vitamin K1, C31H48O2 Synthetic

2-methyl-3-(3,7,11,15-tetramethylhexadecyl)-1, 4-naphthalenedione

a) (VII) K1(25), 2-methyl-3-(3,7,11,15,19-pentamethyl-2- C36H56O2 Synthetic eieosenyl)-1,4-naphthalenedione

(VIII)a) Vitamin K analogs with different Synthetic length of the alkyl side-chain

a) (IX) Phyllohydroquinone, hydroquinone, C31H42O2 Synthetic

dihydrovitamin K1, 2-methyl-3- [(2E,7R, 11R)-3,7,11,15-tetramethylhexadec-2-en-l-yl] naphthalene-1,4-diol a) Internal or external standards.

4. Bibliometric Study phylloquinone) AND TOPIC (HPLC or GC). A similar search was realized with GS tool by also performing an advanced search Searches in the Web of Science Core Collection databases (WoS) for the exact expression in quotation marks throughout the and Google Scholar (GS) web have been done between 1985 and entire document. The methodology of this bibliometric study June 2017 using different keywords and combination of them in and the results of the search strings used in the two sources of the title of the articles. The terms edible, vegetable, or olive have information were synthetized on a graphical representation been used to characterize oils while the synonyms vitamin K1 (Figure 1). The ﬁrst remark was that all of these researches and phylloquinone have been employed to restrict the study only showed that the results were very different from one database to to the compound of interest. This research also focused on PH another. Bibliographic references were more numerous with GS quantiﬁcation methods based on Gas Chromatography (GC) and tool because it provided broader coverage for most disciplines. It High Pressure Liquid Chromatography (HPLC). Searches in was criticized that its coverage was heterogeneous and poorly WoS database have been performed for TOPIC (“edible oil” or informed, including low quality “publications” such as blogs or “vegetable oil” or “olive oil”) AND TOPIC (“vitamin K1” or magazine articles mixing academic and non-academic sources.

GS WoS EO 34 200 4 505 OO 497 000 17 638 VO 244 000 17 763

GS WoS EO & VK 642 4 OO & VK 6 290 13 VO & VK 4 490 21

GS WoS GS WoS GS WoS EO & VK1 71 3 EO & PH 115 4 EO & VK1 & PH 34 2 OO & VK1 671 2 OO & PH 969 7 OO & VK1 & PH 289 2 VO & VK1 422 6 VO & PH 531 18 VO & VK1 & PH 179 4

GS WoS GS WoS GS WoS GS WoS EO & VK1 & HPLC 39 1 EO & VK1 & GC 29 1 EO & VK1 & PH & HPLC 16 1 EO & VK1 & PH & GC 14 1 OO & VK1 & HPLC 241 1 OO & VK1 & GC 188 1 OO & VK1 & PH & HPLC 118 1 OO & VK1 & PH & GC 85 1 VO & VK1 & HPLC 162 1 VO & VK1 & GC 101 1 VO & VK1 & PH & HPLC 87 1 VO & VK1 & PH & GC 51 1

GS WoS GS WoS EO & PH & HPLC 49 3 EO & PH & GC 39 1 OO & PH & HPLC 355 4 OO & PH & GC 251 1 VO & PH & HPLC 207 6 VO & PH & GC 125 2 Websites: Google Scholar (GS); Web of Science (WoS) Topics: EO: “edible oil*”; OO: “olive oil*”; PH: phylloquinone; VO: “vegetable oil*”; VK: “vitamin K”; VK1: “vitamin K1”

Figure 1. Graphical representation of bibliometric search (articles number) on the determination of PH content in vegetable oils. Websites: GS, Google Scholar; WoS, Web of Science. Topics: EO, “edible oil”; OO, “olive oil”; PH, phylloquinone; VO, “vegetable oil”; VK, “vitamin K”; VK1, “vitamin K1”.

Moreover, GS engine found a large number of duplicate papers. (Figure 2), it appeared that the numbers of papers quoting the The typology of publications did not follow the same logic for combined keywords “vitamin K-olive oil” or “vitamin K1-olive WoS model which used a selection of papers and multi- oil” or “phylloquinone-olive oil” increased regularly between disciplinarity, thereby restraining its coverage.[34,35] However, the the years 2000 and 2012, then the interest to study vitamin K has search with the specific term “olive oil” generated a greater grown importantly after the year 2012. The term “phylloqui- number of publications that the global expressions as “edible none” was more used than the expression “vitamin K1” in the and vegetable oil” that it showed the importance of this food different works during these last years. Among the different matrix and the extent of the fields in which olive oil was studied. researches listed in Figure 1, 78 scientific papers showed an Through the combination of the terms relating to oil with the interest for the PH study in different fields (medical, food...), synonyms of vitamin K1, the number of publications decreased including only 25 articles, which treated the PH content in rapidly; it was to highlight that word “phylloquinone” appeared vegetable or edible oils and finally, only 20 publications have more often in the title than “vitamin K1” and some authors used reported results on olive oil specifically. Nevertheless, the olive both terms for more precision. By using an additional search oil data extracted from these 20 publications were often criterion as the analytical technique used for the determination repetitions of some particular studies. It was the case of Booth of phylloquinone content, it appeared that the publications using et al. publications in 1993, 1998, and 2012[3,7,8] and the review of HPLC were more numerous than those dealing with GC. The Eitenmiller et al.[5] Furthermore, Booth and Ferland were co- results found by the WoS model were limited and often identical authors as well as Piironen and Koivu. So, few PH data were with a different combination of keywords. Regarding in details available on olive oil because only 13 scientific articles were – – the publications number found each year from 2000 to 2016 retained.[10 12,15,16,18,19,22,29,36 39] This bibliometric approach

Keywords : "phylloquinone" AND "olive oil*" Keywords: "vita min K" AND "olive oil*" 700 Keywords : "vitamin K1" AND "olive oil*" 120 600 s n s 100 o n i t 500 o i a t c a i l c 80 i b 400 l u b p u f p 60 o 300 f o r e r b e 40 200 b m u m u N

100 N 20

0 0 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 2000 2002 2004 2006 2008 2010 2012 2014 2016 2018 Years Years

Figure 2. Temporal evolution of publication number cited by Google Scholar tool versus used keywords for the bibliometric search. showed that the search using title or keywords converged with realized at about 250 C for refined oils. On the opposite, Fanali difficulty toward the target because the choice of keywords did et al.[41] reported in their review on analytical techniques for fat- not always reflect the publications content. So, among the 119 soluble vitamins that PH was stable to heat and oxygen. Moussa keywords highlighted from the 78 papers, the terms “phyllo- et al.[42] determined PH concentration in intravenous fat quinone” and “vitamin K” were used most often (23 times), emulsions and soybean oils and found variability in PH content “vitamin(s)” for five times, “fat soluble vitamin (s)” for four that they assigned to the nature of the preparation, the producer times, “HPLC” for 10 times, “vegetable oil(s)” for four times and the production batch. PH was extremely sensitive to against two times for “olive oil.” Often, data for edible oils have fluorescent light and daylight and was rapidly destroyed (46–59% been found in publications used general words as “food,”“food and 87–94% loss respectively after 2 days exposure for some composition,” or “food analysis” and “fat soluble vitamins” as vegetable oils)[22] and PH into soybean oil was not detected after keywords. It showed that a correct use of keywords required 48 h of exposure daylight;[42] also it was necessary to work in learning and that all people did not understand the keyword’s subdued light when foods were being analyzed. In addition, E notion in the same way. The difficulty was that the formal (trans-) isomer of PH could be transformed in Z (cis-) isomer treatment of the data by the computers was different of the under light action.[43,44] In addition, PH was unstable in acidic or human way of thinking. alkaline media. So, hot saponification, which can be used for individual and simultaneous extraction of vitamins A, D, and E, [41] is not advisable for vitamin K1. This instability to alkalinity 5. Olive Oil Sampling prohibited the use of saponification for olive oil sample extraction and led to extensive research to develop sample One of the main difficulties encountered in reading the cleanup procedures to overcome insufficiencies of ultra violet published works on olive oils was the lack of precision on the detection for liquid chromatography methodology.[5] A cold geographical or varietal samples origins, their conditioning, saponification have been developed in the dark at room storage conditions, and storage time. However, it was known that temperature,[45,46] using a minimum quantity of alkalis (KOH these parameters had a great influence on the chemical or K CO ) with long reaction time in order to extract different composition of olive oils. In papers relating the PH content, 2 3 nutrients and vitamins (K , MK-4, and MK-7) from animal and olive oils were mainly purchased in local markets without 1 human milk. Extraction yields were between 54% for the MK-4 information on their quality (extra virgin, virgin, or ordinary vitamin and 100% for the other compounds. In addition, Fauler virgin) and their varietal origins. Only the authors, Zakhama et al.[1] pushed further the hypersensitivity of vitamin K towards et al.[38] stated that their sampling were virgin olive oils of the two the strong alkalies by advocating removing traces of detergents main Tunisian varieties (Chemlali and Chetoui), coming from a from washed glasses (intensive wash and glasses heating above continuous industrial production system. In the case of refined 500 C). olive oils, samples were procured from a pool of global suppliers of the food industry.[39]

7. Olive and Vegetable Oil Samples 6. Phylloquinone Stability Preparation and Purification There were few studies that discussed the PH stability in food The low PH content in vegetable oils required a sample matrices when processing samples. PH content quantified in preparation prior to its quantitative analysis to avoid interfer- vegetable oils was relatively stable to processing mode (cold ences with other compounds. The different steps of sample pressed or normal pressed olives)[22] or was degraded slowly in preparation were often done under subdued daylight[19,21,29,37,47] presence of oxygen.[40] It decreased slightly but significantly because the PH was rapidly destroyed by both daylight and (15% loss) after heating at temperatures of 185–190 C[22] but the fluorescent light.[22] The quantitative analysis of PH was PH stability was not known during deodorization process performed using an internal addition standard method as well

Eur. J. Lipid Sci. Technol. 2018, 120, 17005271700527 (5 of 16) © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.ejlst.com as internal or external standard methods. Internal standards the quantity of the phylloquinone added (spiked).[20,36,42,48] 0 0 2 fi (2 ,3 dihydrophylloquinone, menaquinone-4, K1(25), H4-phyllo- Results were dif cult to compare because they were not all quinone) were added to sample prior to its preparation and related to olive oils but to different vegetable oils or margarines whose choice depended on the detection technique. Among and the HPLC detector was not the same. However, the recovery these standard compounds, the most used was 20,30 dihydro- range was 82–100% for the enzymatic method and 85–99% for phylloquinone while PH was used according to a standard the extraction way. With the same experimental conditions for [20,36,42,48] [29] [20] addition method or as an external standard. Gao and the PH detection, the recovery range of trans-vitamin K1 was Ackman[23] advocated an internal standard method that was 93–99% with the extraction method and 82–100% after an much faster and more accurate than an external standard or a enzymatic procedure for a vegetable oil blend (soybean partially standard addition method. In the literature, different extraction hydrogenated and buttermilk). The use of internal standard (K1 0 0 methods have been used to obtain the PH from vegetable oils. In (25);2,3 -dihydrophylloquinone; MK-4) for different oil samples certain experimental procedures, an enzymatic hydroly- and analytical detectors led to a recovery range of 86–103% for an sis[20,23,29,48] or a saponification of oil[38] were performed in a enzymatic process[23,29] and 55–100% for the extraction first step, but more frequently a direct oil solubilization was method.[18,19,21,22,36,37,42] In light of these results, the widely made in organic solvent as hexane[12,18,22,36,37] or solvent mixture used method was the solubilization of oil samples in hexane (acetone/chloroform).[39] For Peterson et al.[18] oils were soluble before purification with a Sep-Pak. This extraction mode, simple, in hexane so no extraction was necessary. The enzymatic rapid, effective, and automatizable, was recommended for digestion was carried on 0.25–0.50 g of oil sample using extracting and concentrating the compound of interest before a 1.00–1.25 g of lipase in 5–100 mL of phosphate buffer. Oil chromatographic analysis. sample was incubated at 37 C with vigorous stirring or in a sonicator bath during 1.5–4 H to ensure complete lipids hydrolysis. Thin-layer chromatography was used once time to 8. Analytical Techniques verify the effectiveness of lipase digestion. The oil saponification (realized with KOH at 80% for 10 min at 80 C) was rarely Various reviews on analysis methods of phylloquinone in food used[38] because of the instability of PH in basic medium.[5,41] have been published in the past and are cited by Fauler et al.[1] Then, oil samples have been generally purified prior to and Etienmiller et al.[5] Thin layer chromatography (TLC) was the chromatographic analysis to remove lipids that may interfere first chromatographic technique used for PH analysis. However, when detecting PH. This purification was often realised on a HPLC has replaced TLC because of its low sensitivity and the Sep-Pak (SPE) silica cartridge[12,18,22,23,39,42,49] and little on difficulty of quantifying small amounts of PH in food matrices. alumina column[48] and sometimes by semi preparative HPLC GC was not used frequently because of low volatility of PH which on μPorasil column.[19,21,37] The SPE extraction with silica required high analysis temperatures (300 C) leading to a cartridge was generally conducted in four steps: (i) a condition- possible degradation of phylloquinone in the chromatographic ing of the stationary phase by a wash of hexane or a mixture or system.[1] To avoid a non-negligible degree of PH degradation hexane/diethyl ether (96:4, v/v); (ii) a loading of the sample during GC analysis, Fauler et al.[1] advocated a reductive acylation frequently dissolved in hexane; (iii) washing (s) of the cartridge in presence of zinc dust, heptafluorobutyric anhydride, and with hexane to eliminate the less polar lipids and the heptafluorobutyric acid in hexane solution. According to the hydrocarbons; and (iv) and finally elution of the fraction works of Osman and Hannestad,[51] an intramolecular rear- containing PH with hexane/diethyl ether (96:4, v/v) or rangement (a shift of the β,γ-double bond on the phytyl side methanol/2-propanol/n-hexane (95:2.5:2.5, v/v/v) or hexane/ chain toward the ring) could be possible when PH was analyzed diisopropyl ether (90:10, v/v). In one case,[18] oil samples were in ethanol solution by GC. further processed by SPE C18 columns. When purification was HPLC was currently the most widely used technique for PH performed with a semi preparative HPLC, a mobile phase with quantification with the help of different detectors allowing a hexane containing 1% diethyl ether was used. In some works, detection of PH or its derivative homologues. Analytical methods the sample purification was performed by washing the hexane available for the PH analysis in olive and vegetable oils were extract with polar solvents (methanol/water or water only).[38,47] resumed in Table 2 and 3 respectively. Most of listed works was However, Cook et al.[20] centrifuged their hexanic sample, which based on a reversed phase HPLC with octadecylsilane-bonded they then filtered through a 0.45 μm glass microfiber filter. C18 phase or much less frequently with triacontyl-bonded C30 Figure 3 summarizes the different steps, identified in the articles phase and seldom on normal-phase HPLC (silica). Little (cited references), for the preparation and the purification of differences were observed on column characteristics (length olive oil samples before their chromatographic analysis. or diameter) and no capillary columns were used as it was done Concerning the validation of these different extraction methods, for some food applications. The interest of using a C30 phase parameters as recovery, precision (repeatability and reproduc- was to make it possible the separation of the PH isomers E and Z ibility), linearity and specificity (peak purity), should have been unlike C18 phase.[20,29] The ability to separate PH isomers and to provided as Kim et al.[50] did it for the determination of PH quantify the E form accurately is very important because E content in legumes and nuts. Few authors specified the recovery isomer is the biological active form of PH while Z isomer is an of their extraction method. When a standard addition method inactive form.[52] Also, E isomer content measured the true was used, the authors made it clear that the recovery was nutritional value of PH.[29] Normal phase HPLC column allowed calculated as the percent difference between the PH quantity to separate E and Z isomers but its implementation was difficult recovered from the spiked and non spiked samples divided by which explained its lack of use in routine analyses.[43,53] Only one

OLIVE OIL SAMPLING Commercial 10-12,16,18,22,36,37,39, extra-virgin 10,19, virgin 38, refined 19,39

INTERNAL STANDARD 11,16,22,29,36,37 2 39 2’-3’-dihydrophylloquinone or H4-phylloquinone or 2-methyl-3-nonadecyl-1,4-naphthoquinone 12 or retinol palmitate 38 or MK-4 19 or 2-methyl-3-(3,7,11,15,19-pentamethyl-2-eicosenyl)-1,4- 18 3 10 naphtalenedione or [1’,2’- H2] phylloquinone (or epoxyde)

SAMPLE PROCESSING Lipase digestion 29 or saponification 38

SAMPLE SOLUBILISATION or EXTRACTION Hexane 12,16,18,19,22,29,36, 37,38 or acetone:chloroform (1:1, v/v) 39 or 2-propanol then hexane 11

PURIFICATION NO PURIFICATION 29 SPE cartridges 10,11,12,16,18,22,37,39 or semi preparative HPLC 10,19 or methanol:water (9:1, v/v) wash 36 or water wash 38

EVAPORATION and/or SOLUBILISATION in mobile phase of chromatographic analysis

EXTERNAL STANDARD CHROMATOGRAPHIC ANALYSES Phylloquinone 29

Figure 3. Different procedures of olive oil sample preparation for the PH quantification. author[39] used a normal phase, a Hypersil silica column to quantification. Careri et al.[54] highlighted this problem by quantify fat-soluble vitamins, including phylloquinone. In his working in vegetable samples. A mobile phase composed with review, Fanali et al.[41] reported that normal phase column could methanol and sodium acetate buffer was used with an tolerate relatively high loads of lipid material, easily removed electrochemical detector operating in the redox mode using from the column by non-polar mobile phase because of their low two electrodes to overcome the drawbacks of one electrode used adsorption. in amperometric detection (oxygen traces, electrode The nature of the mobile phase depended on the detection passivation...). A first electrode reduced phylloquinone in system used: Ultra Violet (UV), electrochemical, fluorescence, phyllohydroquinone while the second one oxidized again the and mass spectrometry. Non-aqueous solvents were used with formed product. An application of this technique for PH UV detection. Three studies reported PH quantification in oils detection in rat liver reported that electrochemical detector was using UV detection[37,38,48] despite the low selectivity and found to be superior in terms of sensibility and selectivity to the sensibility of this detector. Because of the low value of PH UV detector.[55] A comparative study of the sensitivities of a molar absorptivity (e ¼ 19.900 L mol 1 cm 1 at 248 nm),[1] the single carbon electrode cell (reductive mode) and a dual porous detection limits were high with UV detection. In addition, graphite electrode cell (redox mode) was realized to quantify despite the purification of oil sample, residual lipids could show endogenous phylloquinone in plasma and confirmed the an absorbance at the detection wavelength and distorted the PH superiority of the dual-electrode detector.[56] In the case of oil

Table 2. Analytical procedures for PH determination (μg 100 g 1 or μg 100 mL 1) in olive oil samples.

Phylloquinone References Origins N Columns Mobile phase-flow rate Reduction Detector content [8] from [15] Unspecified 1 Unspecified Unspecified Unspecified Unspecified 42 [10] ExtraVirgin 2 Reversed phase C18 Unspecified No Electrochemical redox 74; 85 mode; UV Cheaper oil 1 30 [12] Commercial 1 250 4.6 mm id, CH3OH/CH3CH2OH, Platinum reduction Fluorescence RF-10AXL 63 11 (SD) 5 μm, Capcell Pak C18 95/5 (v/v), 1 mL min 1 column Shimadzu, λex.: 240 nm, UG120, (15 4.0 mm) λem.: 430 nm Shiseido,35 C [16] Commercial 1 150 4.6 mm id, CH3OH, CH3CN, H2O, Postcolumn with dry Fluorescence Hitachi, 65.1 (3.5%CV) 3 μm, C18 Hypersil, 94,5/5/0,5 (v/v/v); isocratic powder Zn L-7480, λex.: 248 nm, λem.: Thermo Scientific (20 3.9 mm) 418 nm

[18] Commercial 2 150 3 mm id, 5 μm, Solvent A:CH3OH þ (10 mM ZnCl2, Postcolumn with dry Fluorescence Shimadzu, 50.1; 70.3; λ λ BDS Hypersil C18, 5mMCH3COOH, 5 mM CH3COONa) powder Zn ex.: 244 nm, em.: 430 nm (14.3) SD; Keystone (1 L); Solvent B: CH2Cl2; gradient (50 2.0 mm, mean 60.2 200 mesh) [19] Italy extra 2 250 4.6 mm id, 95% CH3OH-0,05 CH3COONa, No Electrochemical, dual 44 3(SD); virgin 5 μm, Vydac electrode 50 4(SD); mean 50.0 Refined 2 201 TP54, Hesperia pH3; 1 mL min 1 Analytical cell, ESA 34 2.3 (SD); Coulochem II EC 25.1 0.34 (SD); mean 30.0

[22] Commercial 6 250 4.6 mm id, CH2Cl2 200 mL þ CH3OH Postcolumn with dry Fluorescence Spectroflow 37.2–82.1; μ þ þ 5 m, ODS-Hypersil 800 mL 5 mL (2 M ZnCl2 1M powder Zn 980 Applied Biosystems, mean þ λ λ CH3COOH 1M CH3COONa)/1 L; (20 3.9 mm) ex: 248 nm, em: 418 nm 55.5 6.3 (SD) 1 mL min 1 [29] Unspecified 1 250 4.6 mm id, 3 0.41 g CH3COONa, 1.37 g ZnCl2, 0.30 g Postcolumn with dry Fluorescence RF-2000, E: 80.9; Z: 12.8; μ λ λ Σ and 5 m, C30 YMC, CH3COOH, 920 mL CH3OH, 80 mL powder Zn Dionex, x.: 243 nm, em: 93.7 1 Wilmington CH2Cl2;1–1.5 mL min ; grdient flow (20 4.0 mm) 430 nm [36] μ þ Commercial 1 250 4.6 mm id, 5 m CH2Cl2 100 mL CH3OH 900 mL Postcolumn with dry Fluorescence spectrometer 16.5 (16, 5%

ODS-Hypersil þ (Sol 5 mL 1,37 g ZnCl2 þ 0,30 g powder Zn F-1050, Merck, λex: CV) þ λ Gynkotek, 40 C CH3COOH 0,41g CH3COONa) (20 4.6 mm) 243 nm, em: 430 nm pour 1 L; 1 mL min 1

[37] Commercial 2 300 3.9 mm id, CH3CN, CH2Cl2,CH3OH, 60/20/20 no UV–Vis, Waters 470, 12.7; 18.9; 10 μm, (v/v/v); 1 mL min 1 Millipore, λ: 248 nm mean C18 μ-Bondapak, 15.8 4.4 (SD) Millipore, 20 C [38] – Tunisie 21 250 4.6 mm id, CH3OH, CH3CN, 95/5 (v/v); No UV Vis, Agilent 30.0 5(SD) Virgin 20 4 μm, C18 Hypersil 1 mL min 1 Technologies, λ: 292 nm 40.0 4(SD) Chemlali Virgin Chetoui [39] μ Commercial 14 200 2.1 mm, 1.9 m, Solvent A: n-C6H14 Solvent B: No Xevo TS-Q triple Mean 100 (SD

refined Hypersil GOLDTM 1,4-dioxane, CH3COOH quadrupole tandem MS 100) silica 0,01% v/v, gradient (APCI)

N, sample number; Phylloquinone content (μg 100 g 1 or μg 100 mL 1). samples, two authors used an electrochemical detection in phyllohydroquinone. In the case of electrochemical reduction, [10,19] fl combination with HPLC. Nowadays, uorescence tech- mobile phase was composed with an electrolyte (NaClO4 nique was the most used detection for PH analysis. However, aqueous solutions) and water-miscible organic solvents.[11,42] PH and its homologues did not show native fluorescence and The chemical reduction was performed with a solid phase had to be converted to the corresponding fluorescing reactor (placed between chromatographic column and fluorim- hydroquinone with the help of electrochemical or chemical eter): often full of dry powder zinc and sometimes using postcolumn reduction. The PH derived product obtained was platinum catalyst. It required a mixture of methanol and

Eur. J. Lipid Sci. Technol. 2018, 120, 17005271700527 (8 of 16) © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim u.J ii c.Technol. Sci. Lipid J. Eur. www.advancedsciencenews.com Table 3. Analytical procedures for PH determination in vegetable oil samples.Direct extr., direct extraction; Enzy. dig., enzymatic digestion; N, sample number; Phylloquinone content (mg 100 g 1 or mg 100 mL 1 ).

Varieties Reference Origin No Column Mobile phase-flow rate Reduction Detector Phylloquinone content [22] Almond Commercial 1 250 4.6 mm id, 5 μm, 200 mL CH2Cl2 þ 800 mL CH3OHþ 5mL Postcolumn with Fluorescence Spectroflow 6.70 0.24 (SD) ODS-Hypersil (2 M ZnCl2 þ 1M CH3COOH þ 1M dry powder Zn 980, Applied Biosystems,

2018 1 CH3COONa); 1 mL min (20 3.9 mm) λex: 248 nm, λem.:

, 418 nm 120 [29] Unspecified 1 250 4.6 mm id, 3 and 80 mL CH Cl þ 920 mL CH OH Postcolumn with Fluorescence RF-2000, E:8.7; Z:0.2; Σ:8.9

702 08WLYVHVra mH&C.Ka,Weinheim KGaA, Co. & GmbH Verlag WILEY-VCH 2018 © 1700527 , 2 2 3 5 μm, C30 YMC, (with 0.41 g CH3COONa þ 1.37 g ZnCl2 þ 0.30 g dry powder Zn Dionex, λex.: 243 nm, 1 Wilmington CH3COOH); 1–1.5 mL min ; gradient flow (20 4.0 mm) λem.: 430 nm [29] Avocado Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH (with 0.41 g Postcolumn with Fluorescence RF-2000, E:79.2; Z:0.; Σ:79.2 5 μm, C30 YMC, CH3COONa þ 1.37 g ZnCl2 þ 0.30 g dry powder Zn Dionex, λex.: 243 nm, 1 Wilmington CH3COOH); 1–1.5 mL min ; gradient flow (20 4.0 mm) λem.: 430 nm [11] Corn Commercial 6 Econospher C18 RP, CH3OH þ (CH3)2CHOH þ H2O þ C4H12NB3H8 Electrochemical Fluorescence 2.7–3.1 ; mean 2.9 Alltech (88.5/10/1.5/0.045 (v)); 0.5–1 mL min 1 ; reduction spectrometer Jasco gradient flow 821-FP, λex.: 246 nm, λem.: 430 nm [18] Commercial 2 150 3 mm id, 5 μm, Solvent A:CH3OH þ (10 mM ZnCl2,5mM Postcolumn with Fluorescence Shimadzu, 4.8; 11.1; (14,3 SD); 702 9o 16) of (9 1700527 BDS Hypersil C18, CH3COOH, 5 mM CH3COONa)(1 L); dry powder Zn λex: 244 nm, λem.: mean 7.9 Keystone Solvent B: CH2Cl2; gradient (50 2.0 mm, 430 nm 200 mesh) [20] Commercial Commercial 250 4.6 mm id, 3 μm, 100 mL CH2Cl2 þ 900 mL CH3OH þ 5mL Postcolumn with Fluorescence LS-1, Enzy dig. and extr.: E:1.25 YMC C30 Wilmington (ZnCl2 (50 mM) þ anhydrous sodium acetate dry powder Zn Perkin-Elmer, λex.: (0.3SD); Z:0.8; Direct extr.: (25 mM) þ glacial acetic acid (25 mM)); 243 nm, λem.: 430 nm E:0.95 (0.1 SD); Z:0.4; 0.8 mL min 1 Σ:1.35 [22] Commercial 2 250 4.6 mm id, 5 μm, 200 mL CH2Cl2 þ 800 mL CH3OH þ 5mL Postcolumn with Fluorescence Spectroflow 1.63 0.26(SD) ODS-Hypersil (2M ZnCl2 þ 1M CH3COOH þ 1M dry powder Zn 980, Applied Biosystems, 4,18 0.42 (SD); 1 CH3COONa); 1 mL min (20 3.9 mm) λex.: 248 nm, λem.: mean 2.91 1.28 (SD) 418 nm [36] Corn Commercial 1 250 4.6 mm id, 5 mm 100 mL CH2Cl2 þ 900 mL CH3OH þ 5mL Postcolumn with Fluorescence 1.63 (5.2%CV) ODS Hypersil (1,37 g ZnCl2 þ 0,30 g CH3COOH þ 0,41 g dry powder Zn spectrometer F-1050, 1 Gynkotek, 40 C CH3COONa) pour 1 L; 1 mL min (20 4.6 mm) Merck, λex.: 243 nm, λem.: 430 nm [29] Coconut Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH (with 0.41 g Postcolumn with Fluorescence RF-2000, E:1.5; Z:0.2; Σ:1.7 5 mm, C30 YMC, CH3COONa þ 1.37 g ZnCl2 þ 0.30 g dry powder Zn Dionex, λex.: 243 nm, 1 Wilmington CH3COOH); 1–1.5 mL min ; gradient flow (20 4.0 mm λem.: 430 nm [39] Commercial refined 9 200 2.1 mm, 1.9 mm, Solvent A: n-C6H14 Solvent B: no Xevo TS-Q triple 0 Hypersil GOLDTM 1,4-dioxane þ CH3COOH 0,01% v/v; gradient quadrupole tandem MS silica (APCI) [7]

Cotton Unspecified 1 Unspecified Unspecified Unspecified Unspecified 60 www.ejlst.com seed

(Continued) u.J ii c.Technol. Sci. Lipid J. Eur. www.advancedsciencenews.com Table 3.(Continued)

Varieties Reference Origin No Column Mobile phase-flow rate Reduction Detector Phylloquinone content [22] Palm Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH Postcolumn with Fluorescence RF-2000, E:5.0; Z:1.0; Σ:6.0 5 mm, C30 YMC, (with 0.41 g CH3COONa þ 1.37 g ZnCl2 dry powder Zn Dionex, λex.: 243 nm, 1 Wilmington þ 0.30 g CH3COOH); 1–1.5 mL min (20 4.0 mm λem.: 430 nm

2018 [39] Commercial refined 13 200 2.1 mm, 1.9 mm, Solvent A: n-C6H14 Solvent B: 1,4-dioxane, No Xevo TS-Q triple 0

, Hypersil GOLDTM CH3COOH 0.01% v/v, gradient quadrupole tandem MS 120 silica (APCI) 702 08WLYVHVra mH&C.Ka,Weinheim KGaA, Co. & GmbH Verlag WILEY-VCH 2018 © 1700527 , [22] Peanut Commercial 3 250 4.6 mm id, 5 mm, 200 mL CH2Cl2 þ 800 mL CH3OH þ 5mL Postcolumn with Fluorescence Spectroflow 0.30 0.07–0.47 0.03– ODS Hypersil (2 M ZnCl2 þ 1M CH3COOH þ 1M dry powder Zn 980, Applied Biosystems, 1.19 0.13; 1 CH3COONa)/1 L; 1 mL min (20 3.9 mm) λex.: 248 nm, λem.: mean 0.65 0.27 (SD) 418 nm [29] Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH Postcolumn with Fluorescence RF-2000, E:1.6; Z:0.1; Σ:1.7 5 mm, C30 YMC, (with 0.41 g CH3COONa þ 1.37 g ZnCl2 drypowder Zn Dionex, λex.: 243 nm, 1 Wilmington þ 0.30 g CH3COOH); 1–1.5 mL min ; (20 4.0 mm) λem.: 430 nm gradient flow [36] Pumkin Commercial 1 250 4.6 mm id, 5 mm 100 mL CH2Cl2 þ 900 mL CH3OH þ 5mL Postcolumn with Fluorescence 112 (1.7CV%) ODS Hypersil (1,37 g ZnCl2 þ 0,30g CH3COOH þ 0,41 g dry powder Zn spectrometer F-1050,

702 1 f16) of (10 1700527 1 Gynkotek, 40 C CH3COONa)/1 L; 1 mL min Merck, λex.: 243 nm, λem.: 430 nm Rapeseed [10] Unspecified 2 Unspecified Unspecified No Electrochemical redox 112; 113 or Canola mode; UV [19] Cold pressed 2 250 4.6 mm id, 5 mm, 95% CH3OH/0,05 CH3COONa, No Electrochemical, dual 117 1.1 (SD); 143 1.3 Vydac electrode (SD); mean 130 Commercial refined 2 201 TP54, Hesperia pH3; 1 mL min 1 analytical cell, ESA 140 14 (SD); 160 7 Coulochem II EC (SD); mean 150 [20] Commercial 1 250 4.6 mm id, 3 mm, CH2Cl2 100mL þ CH3OH 900mLþ 5mL Postcolumn with Fluorescence LS-1, Enzy. dig. and extr.:E:107.9 YMC C30 Wilmington, (ZnCl2 (50 mM) þ anhydrous sodium dry powder Zn Perkin-Elmer, λex.: (15.9 SD); Z:9.0 (7.1 SD); NC acetate (25 mM) þ glacial acetic acid 243 nm, λem.: 430 nm Σ:116.9 (25 mM)); 0.8 mL min 1 Commercial 1 [22] Commercial 4 250 4.6 mm id, 5 mm, 200 mL CH2Cl2 þ 800 mL CH3OH þ 5mL Postcolumn with Fluorescence Spectroflow 114.0 14.0–188 2.5; ODS Hypersil (2 M ZnCl2 þ 1M CH3COOH þ 1M dry powder Zn 980, Applied Biosystems, mean 141.0 17.0 (SD) 1 CH3COONa)/1 L; 1 mL min (20 3.9 mm) λex.: 248 nm, λem.: 418 nm [23] Crude undegummed Crude 11111 150 4.6 mm id, 6 mm, 150 mL CH3CN þ 850 mL CH3OH þ 5mL Postcolumn with Fluorescence 420-AC 348 (2.6%CV) 301 (3.7% degummed Commercial refined PartiSphere Whatman CH3CN-CH3OH (15/85 (v/v); 2 M ZnCl2;1M dry powder Zn Waters, λex.: 254 nm, CV) 278 (3.3%CV) 81 1 Expired crude Undegummed C18 CH3COONa; 1 M CH3COOH); 1 mL min (20 3.9 mm) λem.: 400 nm (3.7%CV) 125 (3.2%CV) Commercial

[29] www.ejlst.com Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH (with 0.41 g Postcolumn with Fluorescence RF-2000, E:90.1; Z:21.4; Σ:111.5 5 mm, C30 YMC, CH3COONa þ 1.37 g ZnCl2 þ 0.30 g dry powder Zn Dionex, λex.: 243 nm, 1 Wilmington CH3COOH); 1–1.5 mL min ; gradient flow (20 4.0 mm) λem.: 430 nm

(Continued) u.J ii c.Technol. Sci. Lipid J. Eur. www.advancedsciencenews.com Table 3.(Continued)

Varieties Reference Origin No Column Mobile phase-flow rate Reduction Detector Phylloquinone content [59] Unspecified Unspecified Unspecified Unspecified Unspecified 115–120 from [23] Rapeseed [60] Unspecified / Unspecified Unspecified Unspecified Unspecified 135–306 2018 or Canola from [23]

, [12] μ 120 Commercial 1 250 4.6 mm id, 5 m, CH3OH/CH3CH2OH, 95/5 (v/v), Platinum Fluorescence RF-10Axl 92 25 (SD) 1 λ

702 08WLYVHVra mH&C.Ka,Weinheim KGaA, Co. & GmbH Verlag WILEY-VCH 2018 © 1700527 , Capcell Pak C18 1 mL min reduction column Shimadzu, ex.: 240 nm, UG120, Shiseido, 35 C (15 4.0 mm) λem.: 430 nm [39] Commercial refined 15 200 2.1 mm, 1.9 μm, Solvent A: n-C6H14 Solvent B: 1,4- No Xevo TS-Q triple mean 200 (100SD) Hypersil GOLDTM dioxane, CH3COOH 0,01% v/v, quadrupole tandem MS silica gradient (APCI) [22] Safflower Commercial 2 250 4.6 mm id, 5 μm, 200 mL CH2Cl2 þ 800 mL CH3OH þ 5mL Postcolumn with Fluorescence Spectroflow 6.49.0 0.19–11.77 0.24; ODS- Hypersil (2 M ZnCl2 þ 1M CH3COOH þ 1M dry powder Zn 980, Applied Biosystems, mean 9.03 0.17 (SD) 1 CH3COONa)/1 L; 1 mL min (20 3.9 mm) λex: 248 nm, λem.: 418 nm [29] Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH Postcolumn with Fluorescence RF-2000, E:5.7; Z:0.8; Σ:8.5 5 μm, C30 YMC, (with 0.41g CH3COONa þ 1.37g ZnCl2 þ dry powder Zn Dionex, λex: 243 nm, 702 1 f16) of (11 1700527 1 Wilmington 0.30g CH3COOH); 1–1.5 mL min ; (20 4.0 mm) λem: 430 nm gradient flow [22] Sesame Commercial 2 250 4.6 mm id, 5 pm, 200 mL CH2Cl2 þ 800 mL CH3OH þ 5mL Postcolumn with Fluorescence Spectroflow 12.1.0 1.4–18.7 1.6; ODS-Hypersil (2 M ZnCl2 þ 1M CH3COOH þ 1M dry powder Zn 980, Applied Biosystems, mean 15.5 3.9 (SD) 1 CH3COONa)/1 L; 1 mL min (20 3.9 mm) λex.: 248 nm, λem.: 418 nm Soybean [10] Unspecified 2 Reversed phase C18 Unspecified No Electrochemical redox 112; 150 mode; UV [12] Commercial 1 250 4.6 mm id, 5 μm, CH3OH/CH3CH2OH, 95/5 (v/v), Platinum Fluorescence RF-10Axl 234 48 (SD) Capcell Pak C18 1 mL min 1 reduction column Shimadzu, λex.: 240 nm, UG120, Shiseido, 35 C (15 4.0 mm) λem: 430 nm [19] Commercial refined 1 250 4.6 mm id, 5 μm, 95% CH3OH/0,05 CH3COONa, No Electrochemical, dual 132 1.3 (SD); Vydac 201 TP54, pH3; 1 mL min 1 electrode analytical cell, 158.5 0.13 (SD); mean Hesperia ESA Coulochem II EC 145 [20] Soybean Commercial 1 250 4.6 mm id, 3 mm, 100 mL CH2Cl2 þ 900 mL CH3OH þ ZnCl2 Postcolumn with Fluorescence LS-1, Enzy. dig. and extr.: E:114.2 YMC C30 Wilmington (50 mM) þ anhydrous sodium acetate dry powder Zn Perkin-Elmer, λex.: (7.9SD); Z:20.4 (1.1SD); (25 mM) þ glacial acetic acid (25 mM); 243 nm, λem.: 430 nm Σ:134.6 0.8 mL min 1 Commercial 1 Direct extr.: E:102.5 (3.6SD); Z:17.1 (0,8SD); Σ:119.6

[22] www.ejlst.com Commercial 5 250 4.6 mm id, 5 mm, 200 mL CH2Cl2 þ 800 mL CH3OH þ 5mL Postcolumn with Fluorescence Spectroflow 139.0 4–290 5; ODS Hypersil (2M ZnCl2 þ 1M CH3COOH þ 1M dry powder Zn 980, Applied Biosystems, mean 193 28 (SD) 1 CH3COONa)/ 1 L; 1 mL min (20 3.9 mm) λex.: 248 nm, λem.: 418 nm

(Continued) u.J ii c.Technol. Sci. Lipid J. Eur. www.advancedsciencenews.com Table 3.(Continued)

Varieties Reference Origin No Column Mobile phase-flow rate Reduction Detector Phylloquinone content [23] Commercial 1 150 4.6 mm id, 6 mm, 150 mL CH3CN þ 850 mL CH3OH þ 5mL Postcolumn with Fluorescence 420-AC 250 (2.8%CV) PartiSphere Whatman CH3CN-CH3OH (15/85 (v/v); 2 M ZnCl2; dry powder Zn Waters, λex.: 254 nm, 1 C18 1M CH3COONa; 1 M CH3COOH); 1 mL min λem.: 400 nm

2018 [29] Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH (with 0.41 g Postcolumn with Fluorescence RF-2000, E:236.2; Z:33.6; Σ:269.8 m þ þ λ , 5 m, C30 YMC, CH3COONa 1.37 g ZnCl2 0.30 g dry powder Zn Dionex, ex.: 243 nm, 120 1 Wilmington CH3COOH); 1–1.5 mL min ; gradient flow (20 4.0 mm) λem.: 430 nm 702 08WLYVHVra mH&C.Ka,Weinheim KGaA, Co. & GmbH Verlag WILEY-VCH 2018 © 1700527 , [39] Commercial refined 21 200 2.1 mm, 1.9 mm, Solvent A: n-C6H14 Solvent B: 1,4-dioxane, No Xevo TS-Q triple mean 600 (900SD) Hypersil GOLDTM CH3COOH 0,01% v/v, gradient quadrupole tandem MS silica (APCI) [42] Commercial 3 70 4.7 mm id, 3 mm, CH3CN-C2H5OH, 95/5 (v/v) þ 0.005M Electrochemical Fluorescence linear Fluor 108 (1.7%RSD); 308 1 C18, Beckman NaClO4; 0.8 mL min reduction LC 304, Spectra Physics, (0.9%RSD); 108 λex.: 320 nm, λem.: 430 [48] 1 Commercial 4 150 4.6 mm id, 5 mm, CH3OH-CH3CN-H2O (88/10/2); 1.5 mL min UV–Visible spectrometer 121 12 (SD); 203 11 C18 Supelco Varian, λ: 270 nm (SD); 257 9 (SD); [11] Sunflower Commercial 6 Econospher C18 RP, CH3OH þ (CH3)2CHOH þ H2O þ Electrochemical Fluorescence 5.5–5.9 ; mean 5.7 Alltech C4H12NB3H8 (88.5/10/1.5/0.045 (v)); reduction spectrometer Jasco 702 1 f16) of (12 1700527 0.5–1 mL min 1 ; gradient flow 821-FP, λex.: 246 nm, λem.: 430 nm [19] Sunflower Commercial refined 2 250 4.6 mm id, 5 mm, 95% CH3OH/0,05 CH3COONa, pH3; No Electrochemical, dual 9.2 0.2 (SD); 10 6.1 Vydac 201 TP54, 1 mL min 1 electrode analytical cell, (SD); mean 11.0 Hesperia ESA Coulochem II EC [20] Commercial 1 250 4.6 mm id, 3mm, 100 mL CH2Cl2 þ 900 mL CH3OH þ ZnCl2 Postcolumn with Fluorescence LS-1, Enzy. dig. and extr.: E:1.9 YMC C30 Wilmington (50 mM) þ anhydrous sodium acetate dry powder Zn Perkin-Elmer, λex.: (0.3SD); Z:0.8; Σ:2.7 (25 mM) þ glacial acetic acid (25 mM); 243 nm, λem.: 430 nm 0.8 mL min 1 Commercial 1 Direct extr: E:1.7 (0.35 SD) [22] Commercial 2 250 4.6 mm id, 5 mm, 200 mL CH2Cl2 þ 800 mL CH3OH þ 5mL Postcolumn with Fluorescence Spectroflow 9.19.0 0.79–8.86 0.71; ODSHypersil (2 M ZnCl2 þ 1M CH3COOH þ 1M drypowder Zn 980, Applied Biosystems, mean 9.03 0.17 (SD) 1 CH3COONa)/1 L; 1 mL min (20 3.9 mm) λex.: 248 nm, λem.: 418 nm [29] Unspecified 1 250 4.6 mm id, 3 and 80 mL CH2Cl2 þ 920 mL CH3OH Postcolumn with Fluorescence RF-2000, E 14.9; Z:1.6; Σ:16.5 5 mm, 14.9; Z:1.6; (with 0.41 g CH3COONa þ 1.37 g ZnCl2 dry powder Zn Dionex, λex.: 243 nm, 1 Σ:16.5 C30 YMC, þ 0.30 g CH3COOH); 1–1.5 mL min ; (20 4.0 mm) λem.: 430 nm Wilmington gradient flow [36] Commercial 1 250 4.6 mm id, 5 mm 100 mL CH2Cl2 þ 900 mL CH3OH þ 5mL Postcolumn with Fluorescence 0.97 (0.84CV%) ODSHypersil (1,37 g ZnCl2 þ 0,30 g CH3COOH þ 0,41 g dry powder Zn spectrometer F-1050, 1 Gynkotek, 40 C CH3COONa) pour 1 L; 1 mL min (20 4.6 mm) Merck, λex.: 243 nm, λ em.: 430 nm www.ejlst.com [39] Commercial refined 10 200 2.1 mm, 1.9 mm, Solvent A: n-C6H14 Solvent B: 1,4-dioxane, No Xevo TS-Q triple 0 Hypersil GOLDTM CH3COOH 0,01% v/v, gradient quadrupole tandem MS silica (APCI)

(Continued) www.advancedsciencenews.com www.ejlst.com

dichloromethane in which are added zinc chloride, sodium acetate, and acetic acid to optimize the reduction efﬁciency and the hydrogen gas generation for quinone reduction. The

2 (SD) excitation wavelengths were often comprised between 240 and [23]

15 248 nm; authors used an excitation at 254 nm or worked at 320 nm.[42] The ﬂuorescence emission was measured between 400 and 430 nm. Gao and Ackman[23] tested the repeatability of PH peak height with this derivatization technique and reported that the capacity of a packed column for reduction was excellent

em.: >

λ ( 100 HPLC injections) under their analytical conditions. It was found that the reduction yield of PH was 95% with zinc reduction compared to 60% for electrochemical reduction.[57] 418 nm Only one publication reported the use of mass spectrometry ex.: 248 nm, λ detection for the PH quantiﬁcation in oils using a TS-Q triple 980, Applied Biosystems, Fluorescence Spectroflow quadrupole instrument equipped with an atmospheric pressure chemical ionization probe in positive mode.[39] 3.9 mm) 9. Phylloquinone Content in Olive and (20 dry powder Zn Postcolumn with Vegetable Oils Table 2 and 3 grouped all PH content values extracted from ). 1

literature and measured in olive oils and in 14 vegetable oils used 5mL 1 1M þ for human consummation and in food industry. In most works, þ OH

3 PH content was the sum of E and Z isomers content because of g 100 mL

m the used of a no selective stationary phase (C18). A C30 COOH 3

or stationary phase enabled the separation of E and Z isomers of

1 [20,29]

800 mL CH PH in vegetable oils. In these conditions, E isomer content 1M CH þ 2 þ was always superior to Z isomer proportion. 2 Cl g 100 g 2

m PH content of olive oils reported in the literature ranged from COONa)/ 1 L; 1 mL min

3 12 to 100 μg 100 g 1 in according to the HPLC detection used and CH

(2 M ZnCl the sampling itself for which little information was done, namely

200 mL CH varietal origin, method, and date of manufacture, storage conditions. Furthermore, sample preparation before chemical analyses and detection mode were important elements to take into account in result interpretation. With UV detection, results obtained by two authors[37,38] showed that the PH content was in

4.6 mm id, the ratio of one to two because of the nature of sampling (one was

commercial and the other grouped two certiﬁed cultivars), the m, ODSHypersil

m ﬁ

5 difference in the sample preparation (hot saponi cation or semi preparative HPLC), the divergence in the detection wavelength (at 248 nm, the highest absorbance of PH or at 292 nm, a common wavelength for fat soluble vitamins but a low absorbance of PH,[58] with the incertitude of a no specificPH detection in these analysis conditions). The same coefficient variation (CV) was obtained between the two authors using an UV detector. Fluorescence technique conduced also to PH content with a wide range of variations (37–82 μg 100 g 1), which could be attributed to the same factors cited before and to the Commercial 1 250 derivative technique used to obtain fluorescent compounds. The standard deviation (SD) was of the order of four on average except for two references[12,18] where it was around 11 and 14 for ) one or two samples analyzed. Similar variation ranges for PH contents were obtained with the other detections (electrochemi- [22] cal and MS) but the standard deviation was abnormally high for Continued HPLC-MS analyses (equal to 100 from Nagy et al.[39]). It was .( noted that an extra virgin olive oil seemed to be richer in vitamin K than refined or cheaper oil.[10,19] But this comparison should Wallnut Table 3 Varieties Reference Origin No Column Mobile phase-flow rate Reduction Detector Phylloquinone content Direct extr., direct extraction; Enzy. dig., enzymatic digestion; N, sample number; Phylloquinone content ( be interpreted with caution because it was not the same sample

Min Max

350,0

300,0 ) L m 0 0 1

/ 250,0 g μ r o g 0

0 200,0 1 / g μ ( t n e

t 150,0 n o c e n o n

i 100,0 u q o l l y h

P 50,0

0,0

Varietal origins

*Rapeseed or Canola

Figure 4. Phylloquinone contents of various vegetable oils. perhaps. Only, Woollard et al.[29] quantified E and Z isomers in digestion/extraction or direct extraction). Their results showed olive oils and found that E isomer was 86.3% of the sum of the little significant difference between the two methods. The two isomers. authors concluded that both sample preparations led to the same PH content of the other vegetable oils reported in the precision. In the case of olive oil, only one article published PH literature was a function of varietal origin of oils. Vegetable oils values of E and Z isomers; the content of two E and Z isomers data were reported in function of their commercial importance: was 80.9 and 12.8 μg 100 g 1, respectively.[29] The others works corn, rapeseed (canola), soybean, and sunflower oils are the most mentioned only the total PH content because the used stationary studied. To compare them, the PH extreme values of all oil phase did not separate them. A high standard deviation was samples (extracted from Table 2 and 3) were reported in the often reported with the mean value of PH content because of the Figure 4. Despite of the different analytical techniques used to small number of analyzed samples or a large variability of PH quantify phylloquinone, two oil groups appeared: one describing content of the sampling that it stated by one author working on oils having a maximum PH content superior at 60 μg/100 g numerous oil samples (from 9 to 21 according the varietal (avocado, cottonseed, olive, pumpkin, rapeseed, and soybean) origin).[39] Very few authors provided PH detection limit that and one other closing the oils with a maximum value inferior to varied between 0.01 and 0.5 μg 100 g 1[9,42] for fluorescence 20 μg/100 g. Here the same problematic of oil history resurfaced detection. Test on linearity and sensitivity of LC-dual-electrode or because little information was provided on oil sampling. Studied LC-Fluorescence revealed that the minimum detectable of PH samples were, in the majority of cases, commercial oils that amount was 20–50 pg or 2–10 pg, respectively.[19,29] supposed undergone a refining before their marketing. Only one study[23] reported a decrease of PH content after a transforma- fi tion process (degumming, re ning, bleaching, winterizing, 10. Conclusion deodorizing) of canola oil (fresh or expired) and stored in plastic, metal, or glass bottles but it was not about the same crude oil Bibliometric study showed that few studies related to the used every time. Opposite results were obtained by Piironen determination of phylloquinone content of vegetable oils and in et al.[19] who analyzed two series of Finnish turnip rapeseed oil particular that of olive oils. The published quantification (16 refined and 14 cold pressed oils) without the refined and cold methods were mainly developed for analyzing vegetable, food pressed oils being the same origin. Nagy et al.[39] did not found matrices, and plasma. It appeared that the analytical technique PH in vegetable oils as coconut, palm, and sunflower and they most used to quantify the phylloquinone in vegetable oils was made the assumption that the PH absence was due to the HPLC equipped with a reduction post column coupled with refining process. Nevertheless, other authors who analyzed fluorescence detection. Over time, the sample preparation (the refined oil from the same varietal origin did not obtain a zero most important step of analytic process) has been simplified but value for PH content. Cook et al.[20] compared for the same oil deficiencies continued to exist for the determination of PH and (corn or canola) the impact of the sample preparation (enzymatic internal standards recoveries. In addition, the validation of the

Eur. J. Lipid Sci. Technol. 2018, 120, 17005271700527 (14 of 16) © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.ejlst.com analytical technique was rarely instituted. Most published [10] C. Bolton-Smith, J. G. Rosemary, R. J. G. Price, T. Steven, S. T. Fenton, analyses only measured total vitamin K on a C18 stationary D. J. Harrington, M. J. Shearer, Br. J. Nutr. 2000, 83, 389. phase but a C30 stationary phase allowed the separation of E and [11] L. J. Schurgers, C. Vermeer, Haemostasis 2000, 30, 298. Z PH isomers, to obtain the true nutritional value of PH. This [12] M Kamao, Y. Suhara, N. Tsugawa, M. Uwano, N. Yamaguchi, separation is important because only E isomer is known to have a K. Uenishi, H. Ischida, S. Sasaki, T. Okano, J. Nutr. Sci. Vitaminol. 2007, 53, 464. biological activity. However, the existence of E and Z isomers in [13] G. Ferland, J. A. Sadowski, J. Agric. 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